Recognition of ligand by the a T cell receptor (TCR) is required for the development and maintenance of the T cell repertoire and the initiation and propagation of a cellular immune response. A defining feature of TCRs is their dual capacity for specificity and cross-reactivity. Structural, biophysical, and immunological data continue to provide insight into how TCRs achieve this duality. Yet key questions remain, and a number of hypotheses and generalizations are unproven or untested. The work in this competitive renewal will study the physical bases for TCR specificity and cross-reactivity, focusing on three closely related topics: the dynamics of TCR CDR loops, the mechanisms by which TCRs engage pMHC, and the distribution of binding energy within TCR- pMHC interfaces. Dynamics will be assayed through the use of NMR, time resolved fluorescence anisotropy, and computational simulations, obtaining for the first time clear measurements of TCR loop dynamics across biologically relevant timescales. Measurements of dynamics will be related to TCR specificity and cross- reactivity. Studies of TCR engagement of ligand will be performed with stopped-flow steady-state fluorescence anisotropy, which unlike surface plasmon resonance will allow a rigorous assessment of binding mechanisms (e.g., rigid body, induced fit, or pre-equilibrium conformational ensembles). Again, mechanisms will be related to specificity and cross-reactivity, and considered in the context of popular binding models. In examining the distribution of binding energy, double mutant cycles, an approach commonly used in other fields to asses regional contributions to binding but yet to be applied to TCR-pMHC interactions, will be used. Double mutant cycles will address the relative contributions of various regions within TCR-pMHC interfaces (i.e., the CDR loops, peptide, and MHC helices), as well as examine the extent to which different regions contribute independently of each other. Multiple TCR-pMHC systems will be studied, including cross-reactive interactions, interactions with tight and loose fine specificity, and interactions that proceed with differing degrees of conformational changes and binding topologies. Overall, several outstanding questions regarding TCR recognition of pMHC will be addressed, with the overall aim of clarifying how TCRs achieve their remarkable molecular recognition properties. The results obtained will further the understanding of the normal and abnormal functioning of the human immune system and help in the discovery and design of novel therapeutics based on cellular immunity. Public Health Relevance: A poorly understood feature of T cell receptors of the cellular immune system is their dual capacity for specificity and cross-reactivity. A detailed biophysical study to help understand this duality will be performed, the results of which will contribute not only to the understanding of the normal and abnormal functioning of the human immune system, but also to efforts to discover and design immunologically-based diagnostics and therapeutics.